Sonic hedgehog delivery from self-assembled nanofiber hydrogels reduces the fibrotic response in models of erectile dysfunction

Erectile dysfunction (ED) is a serious medical condition in which current treatments are ineffective in prostatectomy and diabetic patients, due to injury to the cavernous nerve (CN), which causes irreversible remodeling of the penis (decreased smooth muscle and increased collagen), through a largely undefined mechanism. We propose that sonic hedgehog (SHH) and neural innervation, are indispensable regulators of collagen in the penis, with decreased SHH protein being an integral component of the fibrotic response to loss of innervation. We examined collagen abundance and morphology in control (Peyronie’s), prostatectomy and diabetic patients, and in rat models of penile development, CN injury, SHH inhibition and under regenerative conditions, utilizing self-assembling peptide amphiphile (PA) nanofiber hydrogels for SHH delivery. Collagen abundance increased in penis of ED patients. In rats, collagen increased with CN injury in a defined time frame independent of injury severity. An inverse relationship between SHH and collagen abundance was identified; SHH inhibition increased and SHH treatment decreased penile collagen. SHH signaling in the pelvic ganglia (PG)/CN is important to maintain CN integrity and when inhibited, downstream collagen induction occurs. Collagen increased throughout penile development and with age, which is important when considering how to treat fibrosis clinically. These studies show that SHH PA treatment reduces collagen under regenerative post-prostatectomy conditions, indicating broad application for ED prevention in prostatectomy, diabetic and aging patients and in other peripheral nerve injuries. The PA nanofiber protein vehicle may be widely applicable as an in vivo delivery tool

Sonic hedgehog protein is decreased and penile morphology is altered in prostatectomy and diabetic patients.

Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable

(A) Dual TUNEL/α-ACTIN IHC was performed using colorimetric and fluorescent techniques.

<p>Apoptosis was barely detectable in control penile smooth muscle. Apoptosis was abundant in prostatectomy and diabetic corpora cavernosal tissue and almost all apoptosis observed co-localized with α-ACTIN in penile smooth muscle. TUNEL appears in brown (diaminobenzidine, DAB, top) and red (bottom). α-ACTIN appears in red (top) and bluish-purple (bottom). Light blue staining is unavoidable auto-fluorescence present in human penis. Arrows indicate co-localization (shown in pink on bottom). 400× magnification. (B) TUNEL/CD31 IHC shows that apoptosis is also occurring at a very low level in the endothelium. TUNEL appears in brown (DAB) and CD31 appears in red. Arrows indicate co-localization. 400× magnification. Fluorescent co-localization for TUNEL/CD31 was not possible due to the low abundance of endothelium and the high auto-fluorescence present in human penile tissue.</p

Quantification of precursor and active SHH protein by western analysis of rats that underwent bilateral CN crush and sildenafil or DMSO treatment for 7 or 14 days.

<p>Precursor and active SHH proteins were significantly increased 50% and 57% (p = 0.02 and 0.04) after 14 days of sildenafil treatment.</p